2024年4月22日发(作者：怎么联系上门安装宽带)

ialendotoxins

Maizestarch

Sodiumchloride

Agar,accordingtogellingpower

Purifiedwater

1.0g

5.0g

10.0gto15.0g

1000ml

Sterilisebyheatinginanautoclaveat121°Cfor15min.

Ifthesolutionhasinsufficientneutralisingcapacitythe

concentrationofpolysorbate80orlecithinmaybeincreased.

Alternatively,theneutralisersmentionedinTable2.6.13.-3

maybeadded.

Hydratetheagar,dissolvebyheatingtoboilingwith

ssary,adjustthepHsothat

Table2.6.13.-3.–Inactivatorsforantimicrobialagentstobe

aftersterilisationitis7.3±isebyheatinginan

addedtobufferedsodiumchloride-peptonesolutionpH7.0

autoclaveat121°ocoolto45-50°C;

Inactivator

Typeofan-ConcentrationComment

add,wherenecessary,gentamicinsulphatecorrespondingto

timicrobial

20mgofgentamicinbaseandpourintoPetridishes.

agent

SodiumAddafter

Phenolics4g/l

MediumR(Lactosemonohydratesulphitemedium)

Pancreaticdigestofcasein

Yeastextract

Sodiumchloride

Lactosemonohydrate

Cysteinehydrochloride

Purifiedwater

5.0g

2.5g

2.5g

10.0g

0.3g

1000ml

Organo-

mercurals

Halogens

Quaternary

laurilsulfate

Polysorbate80

andlecithin

Eggyolk

Sodium

thioglycolate

Sodium

thiosulphate

Eggyolk

30g/land3g/l

5ml/l-50ml/l

0.5g/l-5g/l

5g/l

5ml/l-50ml/l

Addafter

sterilisationof

bufferedsodium

chloride-peptone

solutionpH7.0

ammonium

sterilisationof

Dissolve,adjusttopH7.1±0.1andfillto8mlin

compounds

bufferedsodium

16mm×160mmtubescontainingasmallDurhamtube.

chloride-peptone

Sterilisebyheatinginanautoclaveat121°Cfor15minand

solutionpH7.0

storeat4°C.

Beforeuse,heatthemediumfor5mininawater-bathand

achtube0.5mlofa12g/lsolutionofsodium

01/2005:20614

metabisulphiteRand0.5mlofa10g/lsolutionofferric

ammoniumcitrate,bothsolutionsbeingfreshlyprepared

IALENDOTOXINS

andfilteredthroughmembranes(poresize:0.45µm).

AgarmediumS(R2A)

Thetestforbacterialendotoxinsisusedtodetector

Yeastextract

0.5g

quantifyendotoxinsofgram-negativebacterialorigin

usingamoebocytelysatefromhorseshoecrab(Limulus

Proteosepeptone

0.5g

polyphemusorTachypleustridentatus).Thereare

0.5g

3techniquesforthistest:thegel-clottechnique,which

Caseinhydrolysate

isbasedongelformation;theturbidimetrictechnique,

0.5g

Glucose

basedonthedevelopmentofturbidityaftercleavageofan

0.5g

endogenoussubstrate;andthechromogenictechnique,

Starch

0.3g

basedonthedevelopmentofcolouraftercleavageofa

Dipotassiumhydrogenphosphate

syntheticpeptide-chromogencomplex.

0.024g

Magnesiumsulphate,anhydrous

Thefollowing6methodsaredescribedinthepresent

0.3g

chapter:

Sodiumpyruvate

Agar

Purifiedwater

15.0g

1000ml

MethodA.

Gel-clotmethod:limittest

MethodB.

Gel-clotmethod:semi-quantitativetest

MethodC.

Turbidimetrickineticmethod

genickineticmethod

genicend-pointmethod

MethodF.

Turbidimetricend-pointmethod

vent

ofdoubtordispute,thefinaldecisionismadebasedupon

methodAunlessotherwiseindicatedinthemonograph.

Thetestiscarriedoutinamannerthatavoidsendotoxin

contamination.

Apparatus

Depyrogenateallglasswareandotherheat-stableapparatus

nlyused

minimumtimeandtemperatureis30minutesat250°

employingplasticapparatus,suchasmicrotitreplatesand

pipettetipsforautomaticpipetters,useapparatusshown

tobefreeofdetectableendotoxinandofinterferingeffects

forthetest.

NOTE:Inthischapter,theterm‘tube’includesalltypesof

receptacles,forexamplemicrotitreplatewells.

161

AdjustthepHsothataftersterilisationitis7.2±0.2.

Sterilisebyheatinginanautoclaveat121°Cfor15min.

NEUTRALISINGAGENTS

Neutralisingagentsmaybeusedtoneutralisetheactivity

ybeaddedtobuffered

sodiumchloride-peptonesolutionpH7.0,preferablybefore

isedtheirefficacyandnon-toxicitytowards

micro-organismsaredemonstrated.

Atypicalneutralisingfluidhasthefollowingcomposition:

Polysorbate80

Lecithin(egg)

Histidinehydrochloride

Peptone(meatorcasein)

Sodiumchloride

Potassiumdihydrogenphosphate

Disodiumhydrogenphosphatedihydrate

Purifiedwater

30g

3g

1g

1g

4.3g

3.6g

7.2g

1000ml

GeneralNotices(1)applytoallmonographsandothertexts

ialendotoxinsEUROPEANPHARMACOPOEIA5.0

Preparationofthestandardendotoxinstocksolution

Thestandardendotoxinstocksolutionispreparedfrom

anendotoxinreferencestandardthathasbeencalibrated

againsttheInternationalStandard,forexampleendotoxin

standardBRP.

EndotoxinisexpressedinInternationalUnits(IU).The

equivalenceinIUoftheInternationalStandardisstatedby

theWorldHealthOrganisation.

NOTE:OneInternationalUnit(IU)ofendotoxinisequalto

oneEndotoxinUnit(E.U.).

Followthespecificationsinthepackageleafletandonthe

labelforpreparationandstorageofthestandardendotoxin

stocksolution.

Preparationofthestandardendotoxinsolutions

Aftervigorouslymixingthestandardendotoxinstock

solution,prepareappropriateserialdilutionsofthissolution

usingwaterforbacterialendotoxinstest(waterforBET).

Usethesolutionsassoonaspossibletoavoidlossofactivity

byadsorption.

Preparationofthetestsolutions

Preparethetestsolutionsbydissolvingordilutingactive

substancesormedicinalproductsusingwaterforBET.

Somesubstancesorpreparationsmaybemoreappropriately

ssary,

adjustthepHofthetestsolution(ordilutionthereof)sothat

thepHofthemixtureofthelysateandtestsolutionfalls

withinthepHrangespecifiedbythelysatemanufacturer.

ThisusuallyappliestoaproductwithapHintherangeof6.0

aybeadjustedbytheuseofacid,baseora

suitablebuffer,asrecommendedbythelysatemanufacturer.

Acidsandbasesmaybepreparedfromconcentratesor

solidswithwaterforBETincontainersfreeofdetectable

smustbevalidatedtobefreeofdetectable

endotoxinandinterferingfactors.

DeterminationoftheMaximumValidDilution

TheMaximumValidDilution(MVD)isthemaximum

allowabledilutionofasampleatwhichtheendotoxinlimit

inetheMVDusingthefollowing

formulae:

—inml/mliftheendotoxinlimitisspecifiedbyvolume

(IU/ml).

λ

=

thelabelledlysatesensitivityinthegel-clot

technique(IU/ml)orthelowestpointused

inthestandardcurveoftheturbidimetricor

chromogenictechniques.

Endotoxinlimit:theendotoxinlimitforactivesubstances

administeredparenterally,definedonthebasisofdose,is

equalto:

GEL-CLOTTECHNIQUE(METHODSAANDB)

Thegel-clottechniqueallowsdetectionorquantification

ofendotoxinsandisbasedonclottingofthelysateinthe

centrationofendotoxins

requiredtocausethelysatetoclotunderstandardconditions

reboththeprecision

andvalidityofthetest,confirmthelabelledlysatesensitivity

andperformthetestforinterferingfactorsasdescribed

atorytesting.

ATORYTESTING

(i)Confirmationofthelabelledlysatesensitivity

Confirmin4replicatesthelabelledsensitivityλ,expressed

inIU/ml,ofthelysatesolutionpriortouseinthetest.

Confirmationofthelysatesensitivityiscarriedoutwhena

newbatchoflysateisusedorwhenthereisanychangein

theexperimentalconditionswhichmayaffecttheoutcome

ofthetest.

Preparestandardsolutionsofatleast4concentrations

equivalentto2λ,λ,0.5λand0.25λbydilutingthestandard

endotoxinstocksolutionwithwaterforBET.

Mixavolumeofthelysatesolutionwithanequalvolume

of1ofthestandardsolutions(suchas0.1mlaliquots)in

ngletestvialsorampoulescontaining

lyophilisedlysateareemployed,addsolutionsdirectlyto

tethereactionmixturefora

constantperiodaccordingtotherecommendationsofthe

lysatemanufacturer(usuallyat37±1°Cfor60±2min),

eintegrityofthegel:fortubes,

takeeachtubeinturndirectlyfromtheincubatorandinvert

itthroughapproximately180°

firmgelhasformedthatremainsinplaceuponinversion,

tisnegativeifanintact

gelisnotformed.

Thetestisnotvalidunlessthelowestconcentrationofthe

standardsolutionsshowsanegativeresultinallreplicate

tests.

Theend-pointisthelastpositiveresultintheseriesof

atethemean

valueofthelogarithmsoftheend-pointconcentrationsand

thentheantilogarithmofthemeanvalueusingthefollowing

expression:

Geometricmeanend-pointconcentration=

K

M

=

=

thresholdpyrogenicdoseofendotoxinper

kilogramofbodymassinasinglehourperiod,

maximumrecommendeddoseofproductper

kilogramofbodymassinasinglehourperiod.

f

=

=

sumofthelogend-pointconcentrationsofthe

dilutionseriesused,

numberofreplicates.

Theendotoxinlimitforactivesubstances

administeredparenterallyisspecifiedinunitssuch

asIU/ml,IU/mg,IU/Unitofbiologicalactivity,etc.,in

monographs.

Concentrationoftestsolution:

—inmg/mliftheendotoxinlimitisspecifiedbymass

(IU/mg),

—inUnits/mliftheendotoxinlimitisspecifiedbyunitof

biologicalactivity(IU/Unit),

162

Thegeometricmeanend-pointconcentrationisthemeasured

sensitivityofthelysatesolution(IU/ml).Ifthisisnotless

than0.5λandnotmorethan2λ,thelabelledsensitivityis

confirmedandisusedinthetestsperformedwiththislysate.

(ii)Testforinterferingfactors

PreparesolutionsA,B,CandDasshowninTable2.6.14.-1,

andusethetestsolutionsatadilutionlessthantheMVD,

notcontaininganydetectableendotoxins,operatingas

atorytesting,(i)Confirmationof

thelabelledlysatesensitivity.

Seetheinformationsectionongeneralmonographs(coverpages)